Articles

https://doi.org/10.1038/s41477-019-0588-4

The hornwort genome and early land plant

evolution
Jian Zhang1,18, Xin-Xing Fu1,2,18, Rui-Qi Li1,18, Xiang Zhao3,18, Yang Liu4,5,18, Ming-He Li6,18,
Arthur Zwaenepoel7,8,18, Hong Ma   9, Bernard Goffinet   10, Yan-Long Guan11, Jia-Yu Xue12,
Yi-Ying Liao4,13, Qing-Feng Wang   13, Qing-Hua Wang1, Jie-Yu Wang6,14, Guo-Qiang Zhang   6,
Zhi-Wen Wang3, Yu Jia1, Mei-Zhi Wang1, Shan-Shan Dong4, Jian-Fen Yang4, Yuan-Nian Jiao   1,
Ya-Long Guo   1, Hong-Zhi Kong   1, An-Ming Lu1, Huan-Ming Yang5, Shou-Zhou Zhang   4,19*,
Yves Van de Peer   7,8,15,16,19*, Zhong-Jian Liu   6,14,17,19* and Zhi-Duan Chen   1,13,19*

Hornworts, liverworts and mosses are three early diverging clades of land plants, and together comprise the bryophytes. Here,
we report the draft genome sequence of the hornwort Anthoceros angustus. Phylogenomic inferences confirm the monophyly of
bryophytes, with hornworts sister to liverworts and mosses. The simple morphology of hornworts correlates with low genetic
redundancy in plant body plan, while the basic transcriptional regulation toolkit for plant development has already been estab-
lished in this early land plant lineage. Although the Anthoceros genome is small and characterized by minimal redundancy,
expansions are observed in gene families related to RNA editing, UV protection and desiccation tolerance. The genome of
A. angustus bears the signatures of horizontally transferred genes from bacteria and fungi, in particular of genes operating in
stress-response and metabolic pathways. Our study provides insight into the unique features of hornworts and their molecular
adaptations to live on land.

L
and plants (Embryophyta) probably originated in the early
Palaeozoic1, initiating the colonization of the terrestrial habi-
tat. Because bryophytes (hornworts, liverworts and mosses)
emerged from the early split in the diversification of land plants, they
are key to the study of early land plant evolution (Supplementary
Note 1.1). Unlike other extant land plants, the vegetative body of
bryophytes is the haploid gametophyte, the sporophyte is always
unbranched and permanently attached to the maternal plant, and
both generations lack lignified vascular tissue2. Bryophytes occur in
nearly all terrestrial habitats on all continents but are absent from
marine environments3.
With only 200–250 species worldwide, the diversity of hornworts
is much lower than that of the other six extant lineages of embryo-
phytes (angiosperms, gymnosperms, ferns, lycophytes, mosses and
liverworts)4. Long considered sister to all other land plants, or sister
to all extant vascular plants, hornworts have recently been resolved
as sister to the setaphytes (that is, the mosses and liverworts) within
monophyletic bryophytes1,5–8. Still, hornworts possess a series of dis-
tinct features9. For instance, most hornworts have chloroplasts with
CO2-concentrating pyrenoids, which have not been found in any
other land plants but are widespread among green algae10. Other
unusual features of hornworts include the persistent basal meristem
in the sporophyte and mucilage-filled cavities for colonial symbi-
onts on the gametophyte11. Most hornworts form tight symbiotic
relationships with cyanobacteria12 and fungal endophytes (espe-
cially Glomeromycota and Mucoromycotina)13.
Here, we present the draft genome of A. angustus Steph.
(Anthocerotaceae) (see Methods, Supplementary Figs. 1 and 2, and
Supplementary Note 1.2). Completion of this high-quality horn-
wort genome complements previously sequenced representatives
of the mosses (Physcomitrella patens14) and liverworts (Marchantia
polymorpha15) and provides a unique opportunity to revisit bryo-
phyte phylogeny, early land plant evolution and the adaptation of
plants to live on land.
Genome assembly and annotation
We sequenced the genome of A. angustus (a single individual of
unknown sex from the dioecious species) using a combination

1
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, China. 2University of Chinese Academy
of Sciences, Beijing, China. 3PubBio-Tech Services Corporation, Wuhan, China. 4Key Laboratory of Southern Subtropical Plant Diversity, Fairy Lake Botanical
Garden, Shenzhen & Chinese Academy of Science, Shenzhen, China. 5BGI-Shenzhen, Shenzhen, China. 6Key Laboratory of National Forestry and Grassland
Administration for Orchid Conservation and Utilization at College of Landscape Architecture, Fujian Agriculture and Forestry University, Fuzhou, China.
7
Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium. 8VIB Center for Plant Systems Biology, Ghent, Belgium. 9Department
of Biology, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA. 10Department of Ecology and Evolutionary Biology,
University of Connecticut, Storrs, CT, USA. 11Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming, China. 12Center for Plant Diversity and Systematics, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, China.
13
Sino–Africa Joint Research Center, Chinese Academy of Sciences, Wuhan, China. 14College of Forestry and Landscape Architecture, South China Agricultural
University, Guangzhou, China. 15Center for Microbial Ecology and Genomics, Department of Biochemistry, Genetics and Microbiology, Pretoria, South Africa.
16
College of Horticulture, Nanjing Agricultural University, Nanjing, China. 17Fujian Colleges and Universities Engineering Research Institute of Conservation and
Utilization of Natural Bioresources, College of Forestry, Fujian Agriculture and Forestry University, Fuzhou, China. 18These authors contributed equally: Jian
Zhang, Xin-Xing Fu, Rui-Qi Li, Xiang Zhao, Yang Liu, Ming-He Li, Arthur Zwaenepoel. 19These authors jointly supervised this work: Shou-Zhou Zhang, Yves Van
de Peer, Zhong-Jian Liu, Zhi-Duan Chen. *e-mail: shouzhouz@126.com; yves.vandepeer@psb.vib-ugent.be; zjliu@fafu.edu.cn; zhiduan@ibcas.ac.cn

Nature Plants | VOL 6 | February 2020 | 107–118 | www.nature.com/natureplants 107

Articles
Table 1 | Assembly and annotation statistics of the draft
genome of A. angustus
Assembly features
 Total length of scaffolds (bp) 119,333,152
 Longest scaffold (bp) 3,809,330
 N50 of scaffold (bp) 1,092,075
 Total length of contigs (bp) 119,122,644
 Longest contig (bp) 3,254,985
 N50 of contig (bp) 796,636
 GC ratio (%) 49.60
Genome annotation
 Number of protein-coding genes 14,629
 Average gene or CDS length (bp) 1,972.11/1,313.24
 Average exon/intron length (bp) 272.63/172.61
 Average exon per gene 4.81
 Average intron per gene 3.81
 Total size of TEs (bp) 72,224,921
 TEs in genome (%) 60.52
CDS, coding sequence.
of Illumina and Oxford Nanopore high-throughput sequencing
systems (see Methods). We generated 126.53 Gb raw reads from
Illumina and 63.61 Gb raw reads from Nanopore sequencing
platforms, and retained 17.10 Gb and 3.78 Gb, respectively, after
filtering, error-correction and decontamination (see Methods,
Supplementary Figs. 2–4 and Supplementary Tables 1–3). Finally,
we obtained an optimized assembly of 119 Mb with a contig N50
length of 796.64 kb and a scaffold N50 length of 1.09 Mb (Table 1
and Supplementary Table 4). Approximately 97.66% of the vegeta-
tive gametophyte transcriptome data for A. angustus genome anno-
tation can be mapped to the assembled genome (Supplementary
Table 5). Repeat sequences comprise 64.21% of the assembled
genome, with transposable elements (TEs) being the major com-
ponent (Table 1 and Supplementary Tables 6 and 7). Among
the TEs, long terminal repeats (LTRs) are the most abundant
(Supplementary Table 7). We used a combination of de  novo,
homology-based and RNA sequence-based predictions to obtain
gene models for the A. angustus genome (Supplementary Table 8).
In total, we predicted 14,629 protein-coding genes with an average
coding-sequence length of 1.31 kb and an average of 4.81 exons per
gene (Table 1, Supplementary Fig. 5 and Supplementary Table 8).
About 85% of these predicted genes have their best hits on plant
sequences from the National Center for Biotechnology Information
(NCBI) non-redundant database (Supplementary Fig. 6),
and 78.39% were functionally annotated through Swissprot,
TrEMBL, Pfam, gene ontology (GO) and Kyoto Encyclopedia of
Genes and Genomes (KEGG) (Supplementary Tables 9 and 32).
Our annotation captured 89.64% of the 956 genes in the BUSCO
plantae dataset16 (85.04% complete gene models plus 4.60% frag-
mented gene models), compared with 93.51% and 92.15% captured
in P. patens14 and M. polymorpha15, respectively (Supplementary
Table 10). In addition to protein-coding genes, we also identified
30 known mature micro RNAs (miRNAs), 180 novel mature miR-
NAs, 347 transfer RNAs, 94 ribosomal RNAs and 83 small nuclear
RNAs (snRNAs) in the A. angustus genome (Supplementary
Table 11). Nine mature miRNA sequences that appear con-
served among land plants (miR156/157, miR159/319, miR160,
miR165/166, miR170/171, miR408, miR477, miR535 and miR536)17
were also found in A. angustus (Supplementary Table 12).
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Comparative genomic analysis
For sequence similarity-based clustering of homologues, we used the
predicted proteomes of A. angustus and 18 other green plants with
fully-sequenced genomes (that is, 11 other land plants, two charo-
phyte green algae and five chlorophyte green algae; Supplementary
Table 13). Genes of A. angustus are distributed among 7,644 gene
families that are shared with other plants, and 497 gene families
that appear to be unique to A. angustus (Fig. 1a and Supplementary
Table 14). In the shared gene families, most A. angustus genes
(that is, 9,680) cluster with land plant genes, and only a very small
number (that is, 107) specifically cluster with green algae genes
(Supplementary Fig. 7). The gene families unique to A. angustus are
enriched in various biosynthetic categories (for example, terpenoid
and zeatin) and various activity categories (for example, nutrient
reservoir activity and catechol oxidase activity) (Supplementary
Tables 15 and 16).
Phylogenetic inferences from 85 single-copy nuclear genes sam-
pled for A. angustus and 18 other green plants resolve hornworts
(A. angustus), mosses (P. patens) and liverworts (M. polymorpha)
as a monophyletic group, with hornworts sister to mosses and
liverworts, which agrees with inferences from 852 nuclear
genes sampled from 103 plant species1 (Fig. 1b, Supplementary
Figs. 8 and 9, Supplementary Table 17 and Supplementary Notes 2.1
and 2.2). The divergence (Supplementary Figs. 10 and 11,
Supplementary Tables 18–20 and Supplementary Note S2.3) of the
extant crown group of hornworts is estimated at 275.62 million
years ago (Ma) (95% highest posterior density, 179.3–384.6 Ma)
(middle Carboniferous–early Jurassic) (Supplementary Fig. 11 and
Supplementary Table 20), which is comparable to the crown age of
hornworts estimated based on two organellar sequences from 77
hornworts and 11 other land plants10. These estimates are thus older
than those inferred from the fossil record, considering that the old-
est putative hornwort fossil is a spore from the Lower Cretaceous
Baqueró Formation, Argentina (from 145 to 100 Ma) that resembles
the spores of extant Anthoceros18.
Comparative genomics shows that the genome of A. angustus
has lost many gene families (that is, 2,145) and comparatively only
modest gains (that is, 497) (Fig. 1b). A similar trend characterizes
the genome of Marchantia and of the ancestor common to all bryo-
phytes, whereas P. patens has gained more families (that is, 1,334)
than it has lost (that is, 1,248; Fig. 1b). Thus, bryophyte genomes
may not only harbour a number of genes and gene families com-
parable to those of vascular plants and in particular seed plants
(Fig. 1b) but may also be highly dynamic through evolutionary time.
Many, if not most, land plants harbour genomic signatures
of ancient whole-genome duplication (WGD)19. However, like
that of Marchantia15, the genome of Anthoceros lacks evidence of
having undergone a WGD (Fig. 1c, Supplementary Fig. 13 and
Supplementary Note 3.1), which confirms the hypothesis drawn
previously from the analysis of transcriptomic data20. The chromo-
somal arrangement of genes is not much conserved among the three
bryophyte lineages (Supplementary Fig. 14a,b and Supplementary
Note 3.2), which likely reflects the ancient divergence of these dif-
ferent lineages of bryophytes. For example, the longest co-linear
block corresponds to a mere five anchor pairs for both A. angustus
versus P. patens and A. angustus versus M. polymorpha, whereas
within the A. angustus genome, the largest co-linear segment con-
sists of six anchor pairs (Supplementary Fig. 14).
The A. angustus genome contains a much lower percentage of
multi-copy gene families than that of single-copy gene families,
implying low genetic redundancy (Supplementary Table 17), which
is similar to what has been observed for the liverwort Marchantia15.
Transcription factors
The A. angustus genome comprises 333 putative transcription fac-
tor (TF) genes covering 61 families, a number that is highly similar
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NATURe PlAnTs Articles
a Setaphyta c 700
Physcomitrella patens
Tracheophyta Physcomitrella patens anchors
2,037 600
Marchantia polymorpha
Anthoceros angustus
844
4 14,652 500
110 Anthoceros angustus anchors

Duplication events
121 390
0 3
363
527 Anthoceros angustus versus Marchantia polymorpha
1099 94 400
238 Anthoceros angustus versus Physcomitrella patens
1,421 3 2
302
42 Marchantia polymorpha versus Physcomitrella patens
4,263 300
911
1 4
48
497
262 33 200
135
5
28
28
18
102
16 18 551
551 1,542 100
73
Anthoceros angustus 80
0
5,440 Charophyta
0 1 2 3 4 5
KS
Chlorophyta
b Species Gene families Orphans Genes
+1,078/–875
Arabidopsis thaliana 12,301 4,206 27,411
+43/–713
12,098
+258/–2,296
+495/–643 Genlisea aurea 10,060 4,434 17,685
Gene families 12,768
gain/loss
+593/–962
Vitis vinifera 12,399 2,172 25,676

+988/-210
12,916 +1,300/–1,170
Oryza sativa 11,950 4,647 27,912
+130/–421
11,820

Tracheophyta
+1,091/–1,271
+127/–932 Phalaenopsis equestris 11,640 9,156 29,431
+1,923/–409 12,111
12,138
+581/–2,222
Zostera marina 10,470 3,888 20,421
+1,228/–710
10,624 +1,092/–1,430
Amborella trichopoda 11,800 8,326 26,846

+376/–580
10,106 +1,689/–3,492
Picea abies 8,821 5,748 26,437

+1,660/–2,181
Selaginella moellendorffii 9,585 4,384 22,273
+2,017/–296
10,310
+1,334/–1,248
Physcomitrella patens 9,566 9,722 35,796
+138/–447 9,480

Bryophyta
+565/–1,101
+497/–370 +238/–759 Marchantia polymorpha 8,944 6,292 19,287
8,589 9,789
+497/–2,145
Anthoceros angustus 8,141 2,997 14,629
+2,042/–0
8,462
+618/–2,955
Chara braunii 6,252 5,220 22,776
Charophyta

+809/–969
Klebsormidium nitens 8,302 3,807 16,044

+391/–780
6,420 Volvox carteri 7,934 3,978 14,434
+2,045/–524
8,323
+639/–293
+179/–845 Chlamydomonas reinhardtii 8,669 5,991 17,741
6,802
Chlorophyta

+467/–1,906
Ulva mutabilis 5,363 5,289 12,924
+1,048/–0
7,468
+251/–1,156
Coccomyxa subellipsoidea 5,850 2,614 9,629
+175/–888 6,755
+245/–1,143
Chlorella variabilis 5,857 2,629 9,780

Fig. 1 | Comparative genomic analysis of A. angustus and 18 other plant species. a, Comparison of the number of gene families identified by OrthoMCL.
The Venn diagram shows the shared and unique gene families in A. angustus, Setaphyta, Tracheophyta, Charophyta and Chlorophyta. The gene-family number
is listed in each of the components. b, Gene-family gain (+)/loss (−) among 19 green plants. The numbers of gained (blue) and lost (red) gene families are
shown above the branches. The boxed number indicates the gene-family size at each node. The number of gene families, orphans (single-copy gene families)
and number of predicted genes is indicated next to each species. c, Comparison of whole paranome, anchor pair and one-to-one orthologue distribution of the
number of synonymous substitutions per synonymous site (KS) across the three bryophyte species (P. patens, M. polymorpha and A. angustus).

to that of the other two bryophyte genomes (Supplementary Fig. 15, of streptophytes and the other with the transition to land15,21. In
Supplementary Table 21 and Supplementary Note 4.1). The diver- plants, genes encoding TFs are among the most highly retained
sity of TF genes in extant plants is rather stable (Supplementary following polyploidy22, a pattern reflected in the comparison of
Fig. 15) and resulted from two ancient bursts of TF families during the three bryophyte genomes14,15. A. angustus and M. polymorpha,
the diversification of green plants: one concomitant with the origin whose genome did not undergo WGDs hold a small number of

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Articles
TF compared to P. patens, which experienced at least one WGD in
its ancestry, resulting in a substantially larger number of TF genes
(Supplementary Fig. 15). It supports the hypothesis that the WGD is
an important mechanism for expansion of TF families23.
Phylogenetic analyses of 24 gene families contributing to the
development of plant body plans or adaptation to the terrestrial envi-
ronment, including 16 TF gene families24,25 (Fig. 2a, Supplementary
Figs. 16–54, Supplementary Table 22 and Supplementary Note 4.2),
confirm that a considerable number of genes, such as genes involved
in gametophyte or sporophyte development, haploid–diploid tran-
sition, meristem development, filamentous growth, photomorpho-
genesis and auxin signalling (Fig. 2), composed the genetic toolkit
of plants before the conquest of land26. In particular, the TF genes for
filamentous growth and auxin signalling arose in charophyte green
algae27,28 (Fig. 2b), which are thought to be the closest living rela-
tives to extant land plants, implying the preliminary establishment
of relatively more complex body plan in these basal streptophytes
for plant terrestrial adaptation29. Furthermore, a set of genes under-
lying key morphological innovations for terrestrial adaptation prob-
ably evolved along with the colonization of land30,31 (Fig. 2b), such
as SMF and ICE for stomatal development (Supplementary Figs. 29
and 30), APB, CLE and CLV1 for 3D growth (Supplementary
Figs. 36 and 50–52), and VNS for water-conducting-cell develop-
ment (Supplementary Fig. 38). The sporophyte morphology of bryo-
phytes is relatively simple, and many of the genes involved in the
elaborate regulation of embryogenesis32, such as FUS3, LEC1, LEC2,
NF-YA1/9 and NF-YA3/5/6/8 are absent in A. angustus, Marchantia
and Physcomitrella (Fig. 2a and Supplementary Figs. 39–41). The
ABI3 genes that mainly function in embryo maturation and seed
desiccation tolerance in flowering plants are present in bryophytes,
and have roles in desiccation tolerance in their vegetative tissues33.
In A. angustus, most genes involved in the development of plant
body plans have a single copy, and a few A. angustus TF gene fami-
lies even lost a subset of duplicates (Fig. 2a and Supplementary
Figs. 16–52). For example, in the bHLH family, the class I RSL gene
that controls the development of rhizoids and root hairs, thought
to have been important for the colonization of land34, is present in
the A. angustus genome, whereas the class II RSL genes respon-
sible for regulating protonema differentiation in P. patens or root
hair elongation in A. thaliana by auxin35 are absent (Supplementary
Fig. 27 and Supplementary Note 4.2). The lack of class II RSL genes
in A. angustus might be related to the morphological simplifica-
tion of this species with respect to tip-growing filamentous struc-
tures2. For the KNOX genes from the homeobox gene family, the
A. angustus genome retains one class II KNOX gene for haploid-
to-diploid morphological transition36, but lacks class I KNOX genes
(Supplementary Fig. 23), whose activity is necessary for seta exten-
sion in the sporophytes in P. patens37. The absence of this gene might
be linked to the absence of setae in hornworts2. The genome of
A. angustus also holds few type II MIKCC MADS-box, class B ARF,
NCARF and short PIN genes, as a result of gene losses suggested
by our phylogenetic analysis (Supplementary Figs. 17, 42, 45 and
Supplementary Note 4.2). The class II RSL, class B ARF, NCARF and
short PIN genes all have auxin-related functions (Supplementary
Note 4.2). Since these auxin-related genes were consistently lost
in A. angustus, this hornwort species possesses the simplest auxin
molecular toolkit among all investigated land plants so far38. Thus,
like the liverwort M. polymorpha15, A. angustus exhibits low redun-
dancy for genes shaping the plant body plan (Fig. 2b). Such a lim-
ited toolkit may be characteristic of the ancestor to bryophytes and
hence, perhaps, of the earliest land plants with a dominant thalloid
gametophyte, and provide the foundation to explaining the architec-
tural simplicity of these plants. By contrast, the genome of P. patens,
which develops a leafy stem, has the most TF genes involved in
the development of plant body plans among the compared bryo-
phytes (Fig. 2b). Although the genome of A. angustus seems poor
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in genes composing the network underlying the development of its
body plan, the TF gene families linked to responses to terrestrial
environmental stimuli exhibit lineage-specific gene expansions in
A. angustus, namely, the LISCL genes for mycorrhizal signalling in
the GRAS gene family39 (Supplementary Fig. 53) and the clade SIP1
for ABA signalling under water stress in the Trihelix gene family40
(Supplementary Fig. 54).
Gene-family expansion
Besides two TF gene families, the A. angustus genome harbours a
variety of other uniquely expanded gene families (Supplementary
Fig. 55). The genome comprises an very large number of pen-
tatricopeptide repeat (PPR) genes for plant organellar RNA pro-
cessing41, accounting for approximately 7.90% of the predicted
protein-coding genes. The expanded PPR genes are PLS-class PPR
genes (Supplementary Fig. 55, Supplementary Tables 23 and 24
and Supplementary Note 5.1). Most of the PLS-class PPR proteins
in A. angustus were predicted to be localized in the mitochon-
drion or chloroplast (Supplementary Table 24). The expansion of
the PLS-class PPR genes correlates with the large number of RNA
editing sites estimated in the organellar genomes of A. angustus
(Supplementary Table 23). Our findings add further support to
the hypothesis that an increase in the number of both RNA editing
sites and PPR genes (especially the PLS-class PPR) occurred after
the separation of land plants from green algae41,42 (Supplementary
Table 23). The reduced number of PPR genes and absence of RNA
editing in marchantiid liverworts are most probably secondary
losses (Supplementary Table 23), as the organellar RNA editing and
plant-specific extensions of PPR genes were also found in junger-
manniid liverworts43. Through RNA editing, the PPR proteins could
act as ‘repair’ factors that alleviate DNA damage caused by increased
UV exposure in terrestrial environments41. Other stress-response
gene families have also expanded in A. angustus, such as cupin and
cytochrome P450 (CYP) (Supplementary Fig. 55). Two groups of
cupin (PF00190) proteins—that is, monocupins and bicupins—
can be recognized on the basis of the number of cupin domains44.
In A. angustus, the cupin gene family has undergone a signifi-
cant expansion (Supplementary Table 25) such that it comprises
more bicupin genes than any other plant (Fig. 3a, Supplementary
Figs. 56 and 57, Supplementary Table 25 and Supplementary
Note 5.2). Expansion of the cupin gene family in A. angustus resulted
mainly from tandem gene duplications (Fig. 3b,c and Supplementary
Note 5.2). Since bicupins (that is, 11S and 7S seed storage proteins)
are desiccation-tolerant proteins in higher land plants44, the large
number of bicupin genes in A. angustus could indicate adaptation for
coping with drought stress in the terrestrial environment. The large
number of A. angustus-specific monocupin genes are homologous
to the P. patens PpGLP6 gene (XP_001782709.1) (Supplementary
Fig. 57 and Supplementary Note 5.2), which encodes a protein
with manganese-containing extracellular superoxide dismutase
(SOD) activity to respond to oxidative stress in terrestrial environ-
ments45. The CYP genes for primary and secondary metabolism
have also expanded in A. angustus (Supplementary Fig. 55 and
Supplementary Note 5.3). For instance, genes belonging to the sub-
families CYP71 and CYP85 contain 56 and 46 genes, respectively
(Supplementary Figs. 58–61 and Supplementary Tables 26 and 27).
The A. angustus CYP genes were assigned to 28 KEGG pathways,
of which ‘flavonoid 3′-monooxygenase/flavonoid 3′,5′-hydroxy-
lase’ and ‘abscisic acid 8′-hydroxylase’ were the most representative
(Supplementary Table 28). Within the CYP71 gene subfamily, genes
homologous to flavonoid 3′-hydroxylase (monooxygenase) (F3'H)
or flavonoid 3′,5′-hydroxylase (F3′5′H) genes that are involved
in flavonoid biosynthesis46 are highly expanded in A. angustus
(Supplementary Fig. 59 and Supplementary Note 5.3). Because fla-
vonoids have an important role in UV-B protection46, the expan-
sion of flavonoid biosynthesis related genes in A. angustus might
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NATURe PlAnTs Articles
a

Vascular plants

Chlorophytes
Charophytes
Liverworts

Hornworts
Mosses
0 1 2 3 ≥4

S. moellendorffii

M. polymorpha
A. trichopoda

C. reinhardtii
A. angustus
A. thaliana

P. patens

C. braunii

V. carteri
K. nitens
Family Clade Function

MIKCc 39 20 3 6 1 0
3 1 0 1
MADS-box MIKC* 7 2 3 11 1 1
M 63 12 13 7 0 11 0 0 1 1 Gametophyte/sporophyte
TCP-I 13 6 5 4 1 1 1 0 0 0 development
TCP
TCP-II 11 9 5 2 1 1 1 0 0 0
LFY 1 1 1 2 1 1 1 1 0 0
RWP-RK RKD 5 3 2 1 1 1 2 5 5 3
BELL 13 6 2 4 1 1 1 1 1 1 Haploid–diploid transition
KNOX-II 4 3 2 2 1 1 0 1
1 1
Homeobox KNOX-I 4 4 3 3 1 0 1 1
WOX 16 9 9 3 1 6 0 1 0 0 Meristem development
HD-Zip-III 5 3 3 5 1 1 1 1 0 0
RSL-I 2 1 3 2 1 1 0 0 0 0
RSL-II 4 1 2 5 1 0 0 0 0 0 Filamentous growth
bHLH
LRL 5 3 3 2 1 2 1 3 0 0
PIF 15 5 4 4 1 1 1 1 0 0
bZIP HY5 2 2 2 2 1 1 1 1 1 2 Photomorphogenesis
NF-YC NF-YC2/3/9 3 2 3 6 2 2 1 1 1 0
ARF-A 5 5 3 8 1 1 0 0 0 0
B3-ARF ARF-B 15 4 2 4 1 0 0 0 0 0 Auxin signalling
ARF-C 3 4 2 3 1 3 1 0 0 0
SMF 3 3 3 2 1 1 0 0 0 0
bHLH Stomatal development
ICE 2 2 3 3 2 1 0 0 0 0
AP2 euANT/APB 8 5 2 4 1 1 0 0 0 0 3D growth
NAC VNS 13 5 4 8 1 1 0 0 0 0 Water-conducting cell development
ABI3 1 1 5 8 2 1 0 0 0 0
B3-LAV FUS3 1 0 0 0 0 0 0 0 0 0
LEC2 1 1 0 0 0 0 0 0 0 0
Embryogenesis
NF-YB LEC1 2 1 1 0 0 0 0 0 0 0
NF-YA1/9 2 1 0 0 0 0 0 0 0 0
NF-YA
NF-YA3/5/6/8 4 2 0 0 0 0 0 0 0 0

b Charophyte green algae

Single-copy TFs
Green algal Hornworts Gene loss
ancestor (A. angustus) Simple gametophyte
Bryophytes

Liverworts Single-copy TFs

(M. polymorpha) Gene loss
Diploid development Simple gametophyte
Photomorphogenesis
Multicellularity (gametophyte) Dominant
Filamentous growth gametophyte Mosses Multiple-copy TFs
Auxin signalling (P. patens) Complex gametophyte

Multicellular sporophyte
Embryogenesis
3D patterning
Stomata
Move to land Vascular plants

Vasculature
Dominant sporophyte

Fig. 2 | Major TFs for plant body plan and evolutionary innovations within plants. a, Overview for the number of major TFs for plant body plan in ten
green plants. Colour key on the upper left of the heatmap denotes the TF numbers. b, Major innovations in plants and evolutionary features of three
bryophyte lineages.

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Articles NATURe PlAnTs

a
Species No. of bicupins No. of monocupins Total

Klebsormidium nitens 9 15 24

Anthoceros angustus 31 48 79

Marchantia polymorpha 1 104 105

Physcomitrella patens 0 20 20

Selaginella moellendorffii 13 50 63

Picea abies 15 31 46

Amborella trichopoda 8 36 44

Arabidopsis thaliana 10 33 43

Oryza sativa 21 43 64

b Scaffold39 c Scaffold44 Scaffold108 Scaffold34 Scaffold13 Scaffold43

AANG004394
AANG004395

AANG009253
AANG009255
AANG009258
AANG009264
AANG009265
AANG013591
AANG009342
AANG009343

AANG008040
AANG013594

AANG008042
AANG008775
AANG008776
AANG008777

950 990 kb 280 300 kb 20 60 kb 740 760 kb 1,140 1,160 kb 550 630 kb

Scaffold64
160 Scaffold24 Scaffold22
AANG011559 910

AANG006112
AANG006111
AANG011560 AANG006497
AANG011562 AANG006501
AANG011563 950 kb
AANG011564 Scaffold6
240 kb
AANG001908 100
880 900 kb
AANG001910
Scaffold15 AANG001913
AANG005009 660 AANG001914
AANG005010 AANG001916
0.6 AANG001917
AANG005011 180 kb
700 kb

Scaffold93 Scaffold95 0.5

AANG013184 80 AANG013297 240
AANG013185 AANG013300
120 kb AANG013304
AANG013306
320 kb

Fig. 3 | Expansion of cupin gene family in A. angustus. a, A summary of the number of cupin genes from nine species based on a Pfam search of cupin_1
domain (PF00190). b,c, Phylogenetic trees show cupin genes in nine plant genomes: bicupins (b) and monocupins (c). The colour of each branch
corresponds to the background colour for each species in a. The tandem duplicated gene clusters are ordered and shown on scaffolds of the A. angustus
genome. The scale bars in the trees show the number of amino acid substitutions per site.

again represent a molecular adaptation to life in the terrestrial
environment. Among the CYP85 genes, the genes homologous to
abscisic acid 8′-hydroxylase genes involved in abscisic acid catabo-
lism during drought stress response47 are also uniquely abundant in
A. angustus (Supplementary Fig. 60 and Supplementary Note 5.3),
and may account for the high desiccation tolerance of A. angustus.
Like the cupin gene family, many of the above expanded gene fami-
lies occur in tandem arrays (Supplementary Table 29). At least 9.82%
of protein-coding genes in A. angustus form ‘tandem’ clusters in the
genome (Supplementary Table 30 and Supplementary Note 5.4),
compared with only 1% in P. patens14 and 5.9% in M. polymorpha15.
CO2-concentrating mechanism
Hornworts are the only extant land plant lineage harbouring a
pyrenoid-based CO2-concentrating mechanism (CCM) similar
to that of green algae9,48 (Supplementary Note 6.1), for which the
key components have been identified49. To clarify whether the
CCM components of green algae have orthologues in hornworts
and other land plants, we searched the A. angustus genome and
other plant genomes or transcriptomes with reference to the CCM
genes from chlorophyte green algae Chlamydomonas reinhardtii49,50
(Supplementary Figs. 62–71 and Supplementary Note 6.2).
A. angustus and all other green plants harbour orthologues of
CAH1/2 whose expression is modulated by external inorganic car-
bon concentration; of CemA, which maintains stromal pH balance;
of LCI11, which mediates the entry of HCO3− in the thylakoid lumen;
and of RCA1 and RBCS1/2, which regulate CO2 fixation by Rubisco
(Supplementary Figs. 62, 65 and 69–72). By contrast, orthologues
of CCP1/2, which mediate the entry of HCO3- into the chloroplast
stroma and of EPYC1, which regulate CO2 fixation by Rubisco were
only present in chlorophyte green algae (Supplementary Figs. 67
and 72 and Supplementary Note 6.2). The three inorganic car-
bon transporters (HLA3, LCI1 and LCIA-like genes) only occur
in bryophytes and green algae, whereas the A. angustus genome
lacks the related orthologues (Supplementary Figs. 63, 66 and 72
and Supplementary Note 6.2). Unexpectedly, the three kinds of car-
bonic anhydrases (CAH3, CAH9 and LCIB/C), which are essen-
tial components of CCM, are conserved in non-angiosperm land

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NATURe PlAnTs Articles
a b 100 Gloeophyllum trabeum ATCC 11539 XP_007862832.1
Oscillatoria nigro-viridis WP_015174426.1 100 Neolentinus lepideus HHB14362 ss-1 KZT26254.1
72 Phormidium tenue WP_073610939.1 Cyanobacteria 80
Cylindrobasidium torrendii FP15055 ss-10 KIY69979.1 Basidiomycota
54 Nostoc calcicola WP_073644548.1 85
Kwoniella heveanensis BCC8398 OCF36487.1
Variovorax sp. CF079 SDD30019.1
Betaproteobacteria Microbotryum lychnidis-dioicae p1A1 Lamole KDE06708.1
Bordetella bronchiseptica WP_033454394.1
100 Taphrina deformans PYCC 5710 CCG84592.1
77 78 Paenibacillus sp. 453mf SFS53973.1
Firmicutes Protomyces lactucaedebilis ORY86103.1 Ascomycota
Lysinibacillus macroides WP_053996692.1
Stenotrophomonas daejeonensis WP_057642067.1 100 Saitoella complicata NRRL Y-17804 ODQ54499.1
98
Gammaproteobacteria
Pseudoxanthomonas dokdonensis WP_057657067.1 100 Anthocero sangustus AANG003233
59 100 Ensifer sp. WSM1721 WP_026621625.1 97 Anthoceros angustus AANG012156 Hornworts
Sinorhizobium meliloti WP_010969737.1 65 100 Anthoceros angustus AANG003219
Phenylobacterium sp. Root700 WP_056732391.1 Alphaproteobacteria Spizellomyces punctatus DAOM BR117 XP_016611677.1
60 Chytridiomycota
77 Rhizobium oryzae WP_075626925.1 Gaertneriomyces semiglobifer 574073
74 88
100 Methylobacterium sp. 10 WP_027174343.1 Mizuhopecten yessoensis XP_021361660.1
93
Anthoceros angustus AANG004679 Hornworts Metazoa
Amphimedon queenslandica XP_003384342.1
100
79 Hoyosella subflava WP_013806789.1 Phytophthora sojae XP_009525471.1 Stramenopiles
81 Williamsia muralis WP_062796745.1
Actinobacteria 100 Coccomyxa subellipsoidea C-169 XP_005645850.1 Chlorophyta
74 Mycobacterium chelonae group WP_057967264.1
Klebsormidium nitens GAQ80065.1 Charophyta
Rhodococcus WP_015889204.1
99 Actinoplanes sp. N902-109 WP_041832545.1
52 Madurella mycetomatis KXX75071.1
100 Micromonospora lupini WP_007459710.1
Fusarium oxysporum Fo47 EWZ28263.1
Mycobacterium sp. IS-1742WP_059094183.1
Verticillium longisporum CRK16593.1 Fungi 100 Bacteria
100 Trichoderma harzianum KKP05707.1 86 Bacillus bataviensis WP_007087256.1
100 Paenibacillus sp. LC231 WP_071219991.1
Purpureocillium lilacinum XP_018184305.1
Brevibacillus formosus WP_047071110.1
0.3 0.4

c 97 Physcomitrella patens XP_001776763.1

d
100 Umezawaea tangerina WP_106185535.1
Physcomitrella patens XP_001775466.1
100 Physcomitrella patens XP_001763702.1 Mosses Pseudonocardia acaciae WP_028921217.1
100 Physcomitrella patens XP_001756707.1 Thermobifida cellulosilytica WP_068753481.1 Actinobacteria
99
Physcomitrella patens XP_001760573.1 98
100 Anthoceros angustus AANG011893 Hornworts Actinoalloteichu scyanogriseus WP_030105734.1
94 Marchantia polymorpha Mapoly0049s0102.1 Liverworts Amycolatopsis taiwanensis WP_027946725.1
81 Pedobacter sp. Hv1 WP_055132054.1 Liverworts
70 60 Marchantia polymorpha Mapoly0076s0083.1
Filimonas lacunae WP_076381825.1
99 100 Physcomitrella patens XP_001778965.1 Mosses
Arcticibacter eurypsychrophilus WP_069661102.1 Bacteroidetes
88 Chitinophaga sp. CF118 WP_090103349.1 Anthoceros angustus AANG008038 Hornworts
Adhaeribacter aquaticus WP_034256883.1
85 Sphingopyxis sp. H115 WP_058804644.1
99 Domibacillus enclensis WP_045851243.1
54 Bacillus sp. SA1-12 WP_046515157.1 99 Methylopila sp. M107 WP_020178725.1 Alphaproteobacteria
100 Firmicutes
Paenibacillus fonticola WP_019640511.1 Sphingomonas sp. JS21-1 WP_093004013.1
95 Clostridiales bacterium SK-Y3 WP_094551178.1
88 Opitutus terrae WP_012377071.1 100 Vibrio atlanticus WP_065679365.1
100 Lacunisphaera sp. TWA-58 WP_129046218.1 Vibrionales bacterium SWAT-3 WP_008216774.1
Verrucomicrobia 57
72
Verrucomicrobia bacterium IMCC26134 WP_082083254.1
100 64 Photobacterium lipolyticum WP_107281819.1 Gammaproteobacteria
Opitutaceae bacterium EW11 WP_107743163.1 96 99
89 Chara braunii GBG82933.1 Oceanimonas baumannii WP_094277847.1
Charophyta
100 53 Chara braunii GBG84520.1 Nitrincola nitratireducens WP_036506495.1
98 Gallaecimonas pentaromativorans WP_050660479.1
97 100 Desulfotomaculum guttoideum WP_092243774.1
Lacimicrobium alkaliphilum WP_062483900.1 Gammaproteobacteria
Marinimicrobium agarilyticum WP_036188610.1 56 Clostridium sp. ASBs410 WP_025234707.1 Firmicutes
97 Lechevalieria aerocolonigenes WP_045318157.1 Lactobacillus acidophilus WP_003548951.1
55 Actinobacteria bacterium 13_2_20CM_2_71_6 OLB77073.1
100 Actinobacteria 96 Methanococcoides methylutens WP_048193719.1
Kutzneria albida WP_025357233.1 Archaea
Streptomyces sviceus WP_037901984.1 Methanosarcina acetivorans WP_011024203.1
93 Haloferax sp. ATB1 WP_042662379.1
100 0.4
Haloarcula japonica WP_004594381.1 Archaea
Halostagnicola larsenii WP_049954255.1
0.4

Fig. 4 | Phylogenetic affinities of genes horizontally transferred to A. angustus. a, Phylogenetic tree of glyoxalase (PF13468). b, Phylogenetic tree of
NAD-binding dehydrogenase (PF08635). c, Phylogenetic tree of glucuronyl hydrolase (PF07470). d, Phylogenetic tree of DNA methyltransferase
(PF02870 and PF01035). The stars indicate that the Anthoceros sequence or bryophyte sequences formed a monophyletic clade with homologues of
putative HGT donor, reflecting Anthoceros-specific or bryophyte-specific HGT events. Maximum-likelihood bootstrap support values ≥50% are shown
above the branches. Red, hornworts and other bryophytes; cyan, green algae; grey, metazoan; orange, stramenopiles; blue, bacteria; yellow, fungi; purple,
archaea. The homologues from the kingdom other than the one that HGT donors are involved in are used as the outgroup. The scale bars in the trees show
the number of amino acid substitutions per site.

plants and green algae (Supplementary Figs. 62, 64, 68 and 72). The
A. angustus genome retains the orthologues of both LCIB/C and
CAH3 genes, but has no copy of CAH9 (Supplementary Fig. 72).
Besides green algae, the essential CCM components occur in both
hornworts and other non-angiosperm land plants that lack pyre-
noids (Supplementary Fig. 72). It implies that the CCM could be an
ancestral mechanism of CO2 fixation by plants, and pyrenoids for
CCM are homologous between hornworts and green algae, whereas
both CCM components and pyrenoids have undergone multiple
losses in land plants in response to atmospheric changes in terres-
trial environments10,48.
Horizontal gene transfer
Horizontal gene transfer (HGT) from bacteria or fungi has been
reported for both the moss P. patens51 and the liverwort M. polymor-
pha15. Consistent with those observations, the taxonomic distribu-
tion of BLASTP hits following careful phylogenetic analysis and
manual inspection suggested that 19 genes from 14 families origi-
nated from HGTs from either bacteria or fungi (Supplementary
Fig. 6 and Supplementary Note 7.1). Bacterial donors are distrib-
uted among nine families: Actinobacteria (three gene families),
Alphaproteobacteria (two gene families), Bacteroidetes (two gene
families), Firmicutes (one gene family) and Verrucomicrobia

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Articles
(one gene family). Five families were acquired from fungi,
belonging to Ascomycota, Basidiomycota, hornwort-symbiotic
Chytridiomycota or Mucoromycota13 (Fig. 4a,b, Supplementary
Figs. 73–84 and 86 and Supplementary Table 31). The detection
of specific HGT in all three fully sequenced bryophytes is remark-
able, and is probably related to the fact that these organisms form
symbioses with diverse bacteria and fungi, which, together with
the weakly protected tissues in the early developmental stages in
the life cycle of these plants, provide the possibility for HGT51.
In addition, we found that two families originating from HGT
from bacteria are shared by the three bryophyte lineages, and
one originating from a HGT from fungi is shared between horn-
worts and liverworts only (Fig. 4c,d, Supplementary Figs. 85 and
86, Supplementary Table 31 and Supplementary Note 7.2). The
HGT genes mentioned above (SCUO value 0.2127) exhibit a sig-
nificantly more biased codon-usage pattern than non-HGT genes
(SCUO value 0.1595) (Supplementary Fig. 87a), which may be
linked to their higher GC content (57.58%) than non-HGT genes
(53.26%) (Supplementary Fig. 87b).
The HGT-derived genes in A. angustus mainly contribute to
metabolic processes, oxidation–reduction and stress response
(Supplementary Table 31). Some transferred genes related to
carbohydrate metabolism are predicted to encode glucuronyl
(AANG011893) and glycosyl hydrolases (AANG004297) (Fig. 4c,
Supplementary Fig. 79 and Supplementary Table 31), which func-
tion in cell wall synthesis and modification and might extend the
metabolic flexibility of A. angustus in changing environments52.
The Alphaproteobacteria-derived gene AANG004679 encodes
glyoxalase, which is related to drought stress tolerance53 (Fig. 4a).
The Actinobacteria-derived DNA methyltransferase genes that are
present only in the three groups of bryophytes are related to DNA
repair54 (Fig. 4d). The hornworts and liverworts share the fungi-
derived terpene synthase-like (MTPSL) genes (Supplementary
Fig. 85). Terpene synthases are pivotal enzymes for the biosynthesis
of terpenoids, which serve as chemical defences against herbivores
and pathogens55. Some horizontally transferred genes in A. angustus,
such as NAD-binding dehydrogenase (Fig. 4b) and MTPSL genes
(Supplementary Fig. 85), underwent subsequent gene duplications.
The results suggest that the acquisition of foreign genes might have
provided additional means for environmental adaptation during
evolution of the hornwort lineage.
Conclusions
As land pioneers, the three bryophyte groups form a well-sup-
ported monophyletic lineage, with hornworts sister to liverworts
and mosses. The genome of hornwort A. angustus shows no evi-
dence of WGDs and low genetic redundancy for networks under-
lying plant body plan, which may be congruent with an overall
simple body plan. Hornworts have retained the essential compo-
nents of CCM found in green algae in response to the atmospheric
changes in terrestrial environments. Meanwhile, the gene inven-
tory in A. angustus expanded mainly through tandem duplication
and HGT. In particular, the expansion of specific gene families
and the acquisition of foreign genes have provided additional met-
abolic abilities in hornworts that probably facilitated their survival
in a terrestrial environment. Together, our results indicate how the
draft genome of A. angustus provides a useful model for studying
early land plant evolution and the mechanism of plant terrestrial
adaptation.
Methods
Sample preparation and sequencing. The natural populations of A. angustus
Steph. were collected from Jinping County, Yunnan Province, China. The voucher
specimen has been deposited at the herbarium, Institute of Botany, Chinese
Academy of Sciences, Beijing, China with collection number W1879-2010-01-18.
The sporophytes of A. angustus were detached from the gametophytes, sterilized in
10% sodium hypochlorite and subsequently rinsed with distilled water56.
114
NATURe PlAnTs
The sporangium was opened and the spores were homogenized and spread onto
the 1/2 KnopII agar medium57 in Petri dishes (Supplementary Fig. 1b). The culture
temperature was between 21 °C and 25 °C. Spores germinated within a couple of
days, and then the sporelings started to grow. After approximately three to four
weeks, the gametophyte started to grow (Supplementary Fig. 1c,d). Since spores
are aposymbiotic, we did not find the phenomenon of mucilage-filled cavities
colonization by cyanobacteria on the A. angustus gametophyte during the sterile
culture. A gametophyte from a single spore was selected and cultured by asexual
propagation. The tissue yielded from subculture was used for genome and RNA
sequencing. We tried to induce sexual reproduction by dropping the growth
temperature of gametophyte cultures to 10 °C and 16 °C, respectively; however
until now they have not yet produced reproductive organs. Therefore, the
sequenced A. angustus is indeed a single-sex individual, which is sequenced at the
gametophyte phase of its life cycle.
Genomic DNA was isolated using the Plant DNAzol reagent for genomic
DNA extraction (Life Technologies) according to the manufacturer’s protocols.
For whole-genome shotgun sequencing, ten sequencing libraries with insert sizes
ranging from 170 bp to 40 kb were generated (Supplementary Table 1). Sequencing
libraries were constructed using a library construction kit (Illumina). All libraries
were sequenced on the Illumina HiSeq 2000 platform. Raw sequencing reads were
trimmed with Trimmomatic (v.0.33)58. Only high-quality reads with a total length
of 126,532,381,412 bp were used for further analysis (Supplementary Table 1). For
Oxford Nanopore sequencing, we constructed a genomic DNA library using the
ONT 1D ligation sequencing kit (SQK-LSK108) according to the manufacturer’s
instructions. The sequencing used a single 1D flow cell on a PromethION
sequencer (Oxford Nanopore Technologies). A total of 63,614,292,295 bp raw reads
were generated, of which 36,070,452,175 bp were retained for further analysis after
filtering and trimming (Supplementary Table 3).
Total RNA was extracted using the PureLink Plant RNA reagent (Life
Technologies) and further purified using TRIzol reagent (Invitrogen). For
transcriptome sequencing (RNA sequencing), libraries with insert sizes ranging
from 200 bp to 500 bp were constructed using the mRNA-Seq Prep Kit (Illumina)
and then sequenced using the Illumina HiSeq 2000 platform. For small-RNA
sequencing, the library was generated from RNA sample using the Truseq
Small RNA Preparation kit (Illumina) and sequenced on the Illumina
HiSeq 2500 platform.
Decontamination. The GC content versus k-mer frequency distribution pattern
of the Illumina raw reads (Supplementary Table 1) after trimming presented two
large groups: one group with a low k-mer frequency (<50) and a wide GC content
distribution range (median number at 0.7), and the other group with a high k-mer
frequency (60–165) and a concentrated GC content distribution range (median
number at 0.5) (Supplementary Fig. 2a). The BLASTN results against the NCBI
nucleotide database revealed that the former sequences were mainly from a variety
of bacteria and the latter were the real genome sequences of A. angustus. We also
investigated the k-mer distributions of the raw reads from the other two published
hornwort genomic sequences, A. agrestis (accession: ERX714368)59 and
Anthoceros punctatus (accession: SRX538621)60, and found a similar distribution
pattern as that of A. angustus, containing two groups, one for the contaminant
sequences and the other for sequences of the plant itself (Supplementary
Fig. 2c,d). Because external bacterial contaminations from the laboratory cause
A. angustus to turn yellow and die during culturing, and all three Anthoceros species
through axenic cultures still have the same bacterial contamination problems
(Supplementary Fig. 2a,c,d), we infer that these bacterial contaminations are
from symbiotic bacteria of Anthoceros that might accompany spores hiding in the
sterilized sporangium. Furthermore, we performed the DAPI staining analysis61 to
investigate the distribution of symbiotic bacteria in A. angustus. The gametophytes
were stained by 0.2 mg l−1 DAPI (4′,6-diamidino-2-phenylindole dihydrochloride;
Sigma, cat. no. D9564) for five minutes. The stained gametophytes were washed
three times, and then observed using confocal microscopy. The bacterial micro-
colonies were observed on the outer surface, as well as in the intercellular space of
the gametophytes of A. angustus (Supplementary Fig. 3). Based on the GC content
versus k-mer frequency distribution pattern of the Illumina raw reads and the result
of the DAPI staining, we could imagine that there is a certain amount of bacterial
sequences remaining in the genome sequencing data of A. angustus. In order to
isolate them, we performed a series of decontamination steps. After generating the
k-mer frequency, we chose the high-abundance k-mer depth (60–165) and retained
the corresponding reads for further analysis. This treatment yielded filtered reads
with a total length of 17,099,027,576 bp (Supplementary Table 2). The distribution
pattern of GC content versus k-mer frequency of the A. angustus filtered reads is
depicted in Supplementary Fig. 2b, which shows an entire group with a sequencing
depth of approximately 150×. Furthermore, we performed error correction for
filtered Nanopore reads using decontaminated Illumina reads by Nextdenovo
(v.2.0)62, resulting in 9,247,957,448 bp corrected reads (Supplementary Table 3).
Through MEGABLAST against the NCBI nucleotide database,
we further removed 5,463,972,682 bp prokaryotic sequences or organellar
sequences, and finally got 3,783,984,766 clean reads with a sequencing depth of
approximately 35× (Supplementary Table 3). A total of approximately
185× coverage was obtained finally.
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NATURe PlAnTs
Genome size estimation. To estimate the genome size of A. angustus, we used
clean Illumina reads to calculate the k-mer distribution. According to the
Lander–Waterman theory63, the genome size can be determined by dividing the
total number of k-mers by the peak value of the k-mer distribution. Because we
sequenced the haploid gametophyte of A. angustus, only one peak was found in
the k-mer distribution. The total number of k-mers was 14,092,039,150, and the
position of the peak was at 132 (Supplementary Fig. 4). The peak was used as
the expected k-mer depth and substituted into the formula genome size = total
k-mer/expected k-mer depth, and the haploid genome size was estimated to be
106,757,872 bp (Supplementary Fig. 4).
Genome assembly and assessment. The clean Nanopore reads after filtering
and decontamination were assembled with wtdbg-1.2.8. After finishing the pre-
assembly (148 Mb), iterative polishing was conducted using Pilon (v.1.22)64 in
which clean Illumina reads were aligned with the pre-assembled contigs. The
pre-assembled contig sequences were performed with the MEGABLAST search
against the NCBI nucleotide database to further remove prokaryotic sequences or
organellar DNA. A total of approximately 29 Mb of data were removed. Further,
we combined the final pre-assembled contig sequences from Nanopore sequencing
and clean paired-read data from Illumina sequencing into scaffolds using SSPACE
(v.3.0)65 tool (Supplementary Table 4). Genome assembly completeness was
assessed using the plantae database of 956 single-copy orthologues using BUSCO
(v.3)16 with a BLAST threshold E-value of 1 × 10−5 (Supplementary Table 10).
Transcriptome assembly and mapping. We used Trimmomatic58 to remove
adaptors from the raw reads of transcriptome sequences and filter out low-quality
reads before assembly. The resulting high-quality reads were de novo assembled
and annotated using Trinity (v.2.5.1)66. For genes with more than one transcript,
the longest transcript was chosen as the unigene and used to predict open reading
frames (ORFs) using TransDecoder (v.5.0.2) (https://github.com/TransDecoder/
TransDecoder/wiki). Finally, we obtained 39,044 unigenes, 26,805 of which had
predicted ORFs. To extend the validation of genome assembly, the transcriptome
was compared to the reference assembly using BLASTN, with an E-value <1 × 10−5.
Of the 26,805 transcripts (>200 bp), 97.66% were successfully mapped back to the
final assembled genome (Supplementary Table 5).
Repeat prediction. Tandem Repeats Finder (v.4.09)67 was used to search for
tandem repeats in the A. angustus genome. Both homology-based and de novo
approaches were used to search for TEs. In the homology-based approach, we used
RepeatMasker (v.4.1.0)67 and RepeatProteinMask68 with the Repbase69 database
of known repeat sequences to search for the TEs in the A. angustus genome. In
the de novo approach, we used LTR_FINDER (v.1.0.2)70, PILER (v.1.3.4.)71 and
RepeatModeler (v.1.0.3)72 to construct a de novo repeat sequence database for
A. angustus and then used RepeatMasker to search for repeats in the genome. All
the repeats identified by different methods were combined into the final repeat
annotation after removing the redundant repeats. The predicted repeats covered
64.21% of the genome sequence (Supplementary Table 6). The categories of
predicted TEs in the A. angustus genome are summarized in Supplementary Table 7.
Genome annotation. To predict protein-coding genes, three approaches were
used: (1) de novo gene prediction, (2) homology-based prediction, and (3)
RNA-sequencing annotation. For de novo prediction, AUGUSTUS (v.2.5.5)73
and GlimmerHMM (v3.0.1)74 were applied to predict genes. For homology-
based prediction, we mapped the protein sequences of five published green plant
genomes (Arabidopsis thaliana, Selaginella moellendorffii, P. patens, M. polymorpha
and Klebsormidium nitens) onto the A. angustus genome using TBLASTN, with a
threshold E-value of 1 × 10−5, and then used GeneWise (v.2.4.1)75 to predict gene
structures. The de novo set and five homologue-based results were combined by
MAKER (v.1.0)76 to integrate a consensus gene set (Supplementary Table 8). To
supplement and improve the gene set, we aligned the RNA-sequencing data to
the genome using TopHat (v2.1.1)77, and the alignments were used as input for
Cufflinks (v.2.2.1)78 with default parameters. We manually combined the MAKER
gene set and ORFs of transcripts to form the final gene set that contains 14,629
genes (Supplementary Table 8).
The A. angustus predicted genes were aligned against the sequences in NCBI
non-redundant protein database using BLASTP79 (E-value <1 × 10−5). According to
the NCBI taxonomy categories of best BLAST hits, the source of A. angustus genes
were classified (Supplementary Fig. 6). Functional annotation of these predicted
genes was obtained by aligning the protein sequences of these genes against
the sequences in public protein databases using BLASTP79 (E-value <1 × 10−5,
identity >30% and coverage >70%, excluding annotations only characterized as
hypothetical or predicted protein), including, SwissProt80, TrEMBL80, Pfam81, GO82
and KEGG83 (Supplementary Tables 9 and 32).
Identification of non-coding RNA genes. To obtain a reliable profile of
A. angustus miRNAs, we used mapped reads from small-RNA sequencing with
reference to the A. angustus draft genome to search against miRNA sequences
in A. thaliana, Oryza sativa, S. moellendorffii, P. patens and C. reinhardtii from
miRBase (http://www.mirbase.org/) for predicting the known miRNAs. The
mapped reads were also used to identify novel miRNAs using miREvo (v.1.2)84
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Articles
software. The tRNA genes were searched by tRNAscan-SE (v.1.3.1)85. The rRNA
genes were predicted by aligning plant rRNA sequences from NCBI (A. thaliana
and Anthoceros agrestis) to the A. angustus genome by BLASTN. The snRNA genes
were predicted using INFERNAL (v.1.1)86 to search from the Rfam database.
Gene-family identification. To construct the dataset for gene-family clustering,
the protein-coding genes from the genomes of A. angustus and 18 other
green plants were used, including those of seven angiosperms (A. thaliana,
Genlisea aurea, Vitis vinifera, O. sativa, Phalaenopsis equestris, Zostera marina and
Amborella trichopoda), one gymnosperm (Picea abies), one lycophyte
(S. moellendorffii), two bryophytes (moss P. patens and liverwort M. polymorpha),
two charophytes (Chara braunii and K. nitens) and five chlorophytes (Volvox
carteri, Chlamydomonas reinhardtii, Ulva mutabilis, Coccomyxa subellipsoidea and
Chlorella variabilis) (Supplementary Table 13). We chose the longest transcript
to represent each gene and removed mitochondrial and chloroplast genes. After
performing an all-against-all BLASTP search with a threshold E-value of 1 × 10−5,
identity >30% and coverage >30%, orthogroups or putative gene families or
subfamilies were identified using OrthoMCL (v.2.0)87, on the basis of a collection of
397,132 predicted protein-coding genes from the above 19 Viridiplantae genomes.
A 5-way comparison of A. angustus, Setaphyta (M. polymorpha and P. patens),
Tracheophyta (vascular plants) (A. thaliana, V. vinifera, O. sativa, Z. marina,
P. equestris, A. trichopoda, P. abies, G. aurea and S. moellendorffii), Charophyta
(C. braunii and K. nitens) and Chlorophyta (V. carteri, C. reinhardtii, U. mutabilis,
C. subellipsoidea and C. variabilis) is shown in Fig. 1a. For A. angustus-specific gene
families, we conducted GO and KEGG enrichment analyses via an enrichment
pipeline (https://sourceforge.net/projects/enrichmentpipeline/).
Phylogenomics. We extracted 85 single-copy gene families shared by 19
Viridiplantae for phylogenomic analysis (Supplementary Note 2.1). The amino
acid alignments of each single-copy gene family were aligned by MAFFT (v.7)88,
and the nucleotide alignments were generated separately with TranslatorX (v0.9)89
on the basis of the corresponding amino acid translation. The amino acid data,
the complete nucleotide data and the first and second codon positions, as well
as the third codon positions, were concatenated as super-matrices. These data
matrices were used for maximum likelihood phylogenetic analyses by RAxML
(v.7.2.3)90 with the GTR + Γ and JTT models for nucleotide and amino acid data,
respectively. For each analysis, the bootstrap support was estimated based on
300 pseudoreplicates using a GTR + CAT approximation. To estimate the degree
of substitutional saturation for the four concatenated datasets mentioned above
(Supplementary Note 2.2), we plotted the uncorrected p-distances against the
inferred distances using the method described by Forterre and Philippe91. The level
of saturation was estimated by computing the slope of the regression line in the
plot; the shallower the slope, the greater the degree of saturation. The maximum
composite likelihood method was used to calculate the inferred distances for
nucleotide data and Poisson correction was used to calculate the inferred distances
for the amino acid data.
To improve the taxon sampling in bryophytes for divergence time estimation,
the transcriptome sequences of 22 other bryophytes were downloaded from the
1KP database92 (http://www.onekp.com/public_data.html) and used in subsequent
analyses (Supplementary Table 18 and Supplementary Note 2.3). The divergence
time was estimated using the MCMCTree program in the PAML package (v.4.7)93
under the nucleotide general time reversible (GTR) substitution model and with
the independent rate model as the molecular clock model. The Markov chain
Monte Carlo (MCMC) process consists of 500,000 burn-in iterations and 1,500,000
sampling iterations (1 sample per 150 iterations). The same parameters were
executed twice to obtain a stable result. We applied nine node constraints in the
age estimate (Supplementary Fig. 10). The minimum and maximum constraints for
each node are shown in Supplementary Table 19.
Gene-family sizes were inferred from the gene-family profile obtained by the
program OrthoMCL. The minimum ancestral gene families were estimated using
DOLLOP program included in the PHYLIP package (v.3.695)94 to determine gene-
family gain or loss evolutions of gene families. There are 8,141 gene families in
the A. angustus genome, 8,944 in M. polymorpha and 9,566 in P. patens, and 9,789
ancestral families in the ancestral bryophyte lineage (Fig. 1b).
KS distribution and co-linearity analysis. All KS distributions were constructed
using wgd (v.3.0)95 using default settings. The M. polymorpha and P. patens genome
data was acquired from the PLAZA resource96. Pairwise co-linearity analyses
within and between A. angustus, M. polymorpha and P. patens were conducted
using I-ADHoRe 3.097 with the following parameter settings: gap_size = 30,
cluster_gap = 35, q_value = 0.75, prob_cutoff = 0.01, anchor_points = 3, alignment_
method = ‘gg2’, level_2_only = ‘false’, table_type = ‘family’ and multiple_hypothesis_
correction = ‘FDR’. Within-genome co-linearity analyses were based on the
paralogous families inferred with wgd, whereas the between-genome co-linearity
analyses were conducted using gene families inferred with OrthoFinder using
default settings.
Analysis of TFs. We used the genome-wide TF prediction program iTAK (v.1.7)98
(http://bioinfo.bti.cornell.edu/cgi-bin/itak/index.cgi) with default parameters to
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preliminarily identify TFs in the above 19 Viridiplantae (Supplementary Tables 13
and 22). The reconstruction of the ancestral state for the individual TF family was
performed using Mesquite (v.3.51)99 (http://mesquiteproject.org/), and the most
parsimonious assumption was taken.
Phylogenetic analysis of gene families. Generally, HMMER search100 with a
domain profile or BLAST search using known protein sequences from other plants
as queries was performed to retrieve the sequences from the A. angustus genome
(Supplementary Notes 4–6). The results of TF prediction by iTAK98 were used as
references. Multiple sequence alignments were performed using the MAFFT88
program (https://mafft.cbrc.jp/alignment/software/). The maximum-likelihood
phylogenetic trees were implemented with RAxML-HPC2 on XSEDE101 through
the CIPRES Science Gateway (v.3.3) (https://www.phylo.org/), estimating branch
support values by bootstrap iterations with 1,000 replicates.
Gene-family expansion identification. To understand gene-family expansion
or contraction in A. angustus compared with that in 18 other green plants, the
mean gene-family size was calculated for all gene families (excluding orphans
and species-specific families). The number of genes per species for each family
was transformed into a matrix of z-scores to centre and normalize the data. The
first 100 families with the largest gene-family size in A. angustus were selected
(Supplementary Fig. 55). The clustering and visualization were performed using
Genesis (v.3.0)102. The functional annotation of each family was predicted on
the basis of sequence similarity to entries in the Pfam protein domain database,
where more than 30% of proteins in the family share the same protein domain.
Transposon-derived gene families were removed because the distribution of such
families is likely to be a consequence of the gene models derived from a repeat-
masked genome sequence and therefore may be artefactual103.
Tandem duplication definition. Genes were defined as tandemly arrayed genes if
they belonged to the same family, were located within 100 kb each other, and were
separated by zero, one or fewer, five or fewer, or ten or fewer non-homologous
intervening ‘spacer’ genes104. Therefore, the four sets of tandem gene definitions
were analysed.
HGT event identification. In this study, we used two different strategies to identify
candidates for A. angustus-specific and bryophyte-specific HGTs. For A. angustus-
specific HGTs, we submitted 14,629 predicted coding genes of A. angustus to a
BLASTP search against the NCBI protein database (E-value cutoff of 1 × 10−5)
(Supplementary Note 7.1). The proteins with the best BLAST hits in bacterial or
fungal sequences were extracted. After sequences without support of transcript
evidence were excluded, a series of parameters were used to filter the candidates
(Supplementary Note 7.1). For the bryophyte-specific HGT, we extracted gene
families that are common to at least two of the three members of bryophytes (moss
P. patens, liverwort M. polymorpha and hornwort A. angustus). To preliminarily
determine whether these clusters are HGT candidates, we submitted the
corresponding A. angustus members of each cluster to the NCBI protein database
for BLASTP search and checked the taxonomy report of the top 1,000
BLAST hits (Supplementary Note 7.2). The homologues of published HGTs
in P. patens51 and M. polymorpha15 were also investigated in the A. angustus
genome. All candidate HGTs were subjected to phylogenetic analysis for
verification. Synonymous codon-usage order values and GC contents of HGT and
non-HGT genes were calculated by CodonO105.
Reporting Summary. Further information on experimental design is available in
the Nature Research Reporting Summary linked to this article.
Data availability
The A. angustus genome project has been deposited at the NCBI under the
BioProject number PRJNA543716. The genome sequencing data were deposited in
the Sequence Read Archive database under the accession number SRR9696346. The
A. angustus transcriptome project has been deposited at the NCBI under BioProject
PRJNA543724. The transcriptome sequencing data were deposited in the Sequence
Read Archive database under the accession number SRR9662965. The assembled
genome sequences, gene models and miRNA data are available via DRYAD
(https://doi.org/10.5061/dryad.msbcc2ftv). All data that support the findings of
this study are also available from the corresponding authors upon request.
Received: 12 June 2019; Accepted: 20 December 2019;
Published online: 10 February 2020
References
1. Puttick, M. N. et al. The interrelationships of land plants and the nature of
the ancestral embryophyte. Curr. Biol. 28, 733–745 (2018).
2. Goffinet, B. & Buck, W. R. in The Evolution of Plant Form (eds Ambrose, B.
& Purruganan, M.) 51–90 (Wiley–Blackwell, 2013).
3. von Konrat, M., Shaw, A. J. & Renzaglia, K. S. A special issue of Phytotaxa
dedicated to bryophytes: the closest living relatives of early land plants.
Phytotaxa 9, 5–10 (2010).
116
NATURe PlAnTs
4. Christenhusz, M. J. M. & Byng, J. W. The number of known plants species
in the world and its annual increase. Phytotaxa 261, 201–217 (2016).
5. Qiu, Y. L. et al. The deepest divergences in land plants inferred
from phylogenomic evidence. Proc. Natl Acad. Sci. USA 103,
15511–15516 (2006).
6. Wickett, N. J. et al. Phylotranscriptomic analysis of the origin and
early diversification of land plants. Proc. Natl Acad. Sci. USA 111,
E4859–E4868 (2014).
7. Cox, C. J., Li, B., Foster, P. G., Embley, T. M. & Civan, P. Conflicting
phylogenies for early land plants are caused by composition biases among
synonymous substitutions. Syst. Biol. 63, 272–279 (2014).
8. Liu, Y., Cox, C. J., Wang, W. & Goffinet, B. Mitochondrial phylogenomics of
early land plants: mitigating the effects of saturation, compositional
heterogeneity, and codon-usage bias. Syst. Biol. 63, 862–878 (2014).
9. Villarreal, J. C. & Renzaglia, K. S. The hornworts: important advancements
in early land plant evolution. J. Bryol. 37, 157–170 (2015).
10. Villarreal, J. C. & Renner, S. S. Hornwort pyrenoids, carbon-concentrating
structures, evolved and were lost at least five times during the last 100
million years. Proc. Natl Acad. Sci. USA 109, 18873–18878 (2012).
11. Renzaglia, K. S., Villarreal, J. C. & Duff, R. J. in Bryophyte Biology
(eds Goffinet, B. & Shaw, J.) 139–171 (Cambridge Univ. Press, 2009).
12. Adams, D. G. & Duggan, P. S. Cyanobacteria–bryophyte symbioses.
J. Exp. Bot. 59, 1047–1058 (2008).
13. Desirὸ, A., Duckett, J. G., Pressel, S., Villarreal, J. C. & Bidartondo, M. I.
Fungal symbioses in hornworts: a chequered history. Proc. R. Soc. B 280,
1759 (2013).
14. Rensing, S. A. et al. The Physcomitrella genome reveals evolutionary insights
into the conquest of land by plants. Science 319, 64–69 (2008).
15. Bowman, J. L. et al. Insights into land plant evolution garnered from the
Marchantia polymorpha genome. Cell 171, 287–304 (2017).
16. Simao, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. &
Zdobnov, E. M. BUSCO: assessing genome assembly and annotation
completeness with single-copy orthologs. Bioinformatics 31,
3210–3212 (2015).
17. Axtell, M. J. & Bowman, J. L. Evolution of plant microRNAs and their
targets. Trends Plant Sci. 13, 343–349 (2008).
18. Archangelsky, S. & Villar de Seone, L. Estudios palinógicos de la formación
Baqueró (Cretácico), provincia de Santa Cruz, Argentina. Ameghiniana 35,
7–19 (1996).
19. Van de Peer, Y., Mizrachi, E. & Marchal, K. The evolutionary significance of
polyploidy. Nat. Rev. Genet. 18, 411–424 (2017).
20. Lang, D. et al. The Physcomitrella patens chromosome-scale assembly
reveals moss genome structure and evolution. Plant J. 93, 515–533 (2018).
21. Catarino, B., Hetherington, A. J., Emms, D. M., Kelly, S. & Dolan, L. The
stepwise increase in the number of transcription factor families in the
Precambrian predated the diversification of plants on land. Mol. Biol. Evol.
33, 2815–2819 (2016).
22. Cheng, F. et al. Gene retention, fractionation and subgenome differences in
polyploid plants. Nat. Plants 4, 258–268 (2018).
23. Lang, D. et al. Genome-wide phylogenetic comparative analysis of plant
transcriptional regulation: a timeline of loss, gain, expansion, and
correlation with complexity. Genome Biol. Evol. 2, 488–503 (2010).
24. Sakakibara, K. Technological innovations give rise to a new era of plant
evolutionary developmental biology. Adv. Bot. Res. 78, 3–35 (2016).
25. Szövényi, P., Waller, M. & Kirbis, A. Evolution of the plant body plan.
Curr. Top. Dev. Biol. 131, 1–34 (2019).
26. Floyd, S. K. & Bowman, J. L. The ancestral developmental tool kit of land
plants. Int. J. Plant Sci. 168, 1–35 (2007).
27. Hori, K. et al. Klebsormidium flaccidum genome reveals primary factors for
plant terrestrial adaptation. Nat. Commun. 5, 3978 (2014).
28. Nishiyama, T. et al. The Chara genome: secondary complexity and
implications for plant terrestrialization. Cell 174, 448–464 (2018).
29. Wodniok, S. et al. Origin of land plants: do conjugating green algae hold
the key? BMC Evol. Biol. 11, 104 (2011).
30. Ishizaki, K. Evolution of land plants: insights from molecular studies on
basal lineages. Biosci. Biotechnol. Biochem. 81, 73–80 (2017).
31. Rensing, S. A. Great moments in evolution: the conquest of land by plants.
Curr. Opin. Plant Biol. 42, 49–54 (2018).
32. Braybrook, S. A. & Harada, J. J. LECs go crazy in embryo development.
Trends Plant Sci. 13, 624–630 (2008).
33. Takezawa, D., Komatsu, K. & Sakata, Y. ABA in bryophytes: how a
universal growth regulator in life became a plant hormone? J. Plant Res.
124, 437–453 (2011).
34. Proust, H. et al. RSL class I genes controlled the development of
epidermal structures in the common ancestor of land plants. Curr. Biol. 26,
93–99 (2016).
35. Pires, N. D. et al. Recruitment and remodeling of an ancient gene regulatory
network during land plant evolution. Proc. Natl Acad. Sci. USA 110,
9571–9576 (2013).
Nature Plants | VOL 6 | February 2020 | 107–118 | www.nature.com/natureplants
NATURe PlAnTs
36. Sakakibara, K. et al. KNOX2 genes regulate the haploid-to-diploid
morphological transition in land plants. Science 339, 1067–1070 (2013).
37. Coudert, Y., Novák, O. & Harrison, C. J. A KNOX-cytokinin regulatory
module predates the origin of indeterminate vascular plants. Curr. Biol. 29,
2743–2750 (2019).
38. Mutte, S. K. et al. Origin and evolution of the nuclear auxin response system.
eLife 7, e33399 (2018).
39. Cenci, A. & Rouard, M. Evolutionary analyses of GRAS transcription
factors in angiosperms. Front. Plant Sci. 8, 273 (2017).
40. Kaplan-Levy, R. N., Brewer, P. B., Quon, T. & Smyth, D. R. The trihelix
family of transcription factors—light, stress and development. Trends Plant
Sci. 17, 163–171 (2012).
41. Fujii, S. & Small, I. The evolution of RNA editing and pentatricopeptide
repeat genes. N. Phytol. 191, 37–47 (2011).
42. Cheng, S. et al. Redefining the structural motifs that determine RNA
binding and RNA editing by pentatricopeptide repeat proteins in land
plants. Plant J. 85, 532–547 (2016).
43. Rüdinger, M., Polsakiewicz, M. & Knoop, V. Organellar RNA editing and
plant-specific extensions of pentatricopeptide repeat proteins in
jungermanniid but not in marchantiid liverworts. Mol. Biol. Evol. 25,
1405–1414 (2008).
44. Dunwell, J. M., Khuri, S. & Gane, P. J. Microbial relatives of the seed storage
proteins of higher plants: conservation of structure and diversification of
function during evolution of the cupin superfamily. Microbiol. Mol. Biol.
Rev. 64, 153–179 (2000).
45. Nakata, M. et al. Germin-like protein gene family of a moss, Physcomitrella
patens, phylogenetically falls into two characteristic new clades. Plant Mol.
Biol. 56, 381–395 (2004).
46. Pollastri, S. & Tattini, M. Flavonols: old compounds for old roles. Ann. Bot.
108, 1225–1233 (2011).
47. Sakata, Y., Komatsu, K. & Takezawa, D. in Progress in Botany (ed. Lüttge, U.)
57–96 (Springer-Verlag, 2014).
48. Hanson, D. T., Renzaglia, K. & Villareal, J. C. in Photosynthesis of
Bryophytes and Early Land Plants (eds Hanson, D. T. & Rice, S. K.)
95–111 (Springer, 2014).
49. Meyer, M. & Griffiths, H. Origins and diversity of eukaryotic CO2-
concentrating mechanisms: lessons for the future. J. Exp. Bot. 64,
769–786 (2013).
50. Mackinder, L. C. M. A spatial interactome reveals the protein organization
of the algal CO2-concentrating mechanism. Cell 171, 133–147 (2017).
51. Yue, J., Hu, X., Sun, H., Yang, Y. & Huang, J. Widespread impact of
horizontal gene transfer on plant colonization of land. Nat. Commun. 3,
1152 (2012).
52. Foflonker, F. et al. Genome of the halotolerant green alga Picochlorum sp.
reveals strategies for thriving under fluctuating environmental conditions.
Environ. Microbiol. 17, 412–426 (2015).
53. Hasanuzzaman, M. et al. Coordinated actions of glyoxalase and antioxidant
defense systems in conferring abiotic stress tolerance in plants. Int. J. Mol.
Sci. 18, 200 (2017).
54. Finnegan, E. J. & Kovac, K. A. Plant DNA methyltransferases. Plant Mol.
Biol. 43, 189–201 (2000).
55. Jia, Q. et al. Microbial-type terpene synthase genes occur widely in nonseed
land plants, but not in seed plants. Proc. Natl Acad. Sci. USA 113,
12328–12333 (2016).
56. Duckett, J. G. et al. In vitro cultivation of bryophytes: a review of
practicalities, problems, progress and promise. J. Bryol. 26, 3–20 (2004).
57. Kugita, M. et al. The complete nucleotide sequence of the hornwort
(Anthoceros formosae) chloroplast genome: insight into the earliest land
plants. Nucleic Acids Res. 31, 716–721 (2003).
58. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for
Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
59. Szövényi, P. et al. Establishment of Anthoceros agrestis as a model species
for studying the biology of hornworts. BMC Plant Biol. 15, 98 (2015).
60. Li, F. et al. Horizontal transfer of an adaptive chimeric photoreceptor from
bryophytes to ferns. Proc. Natl Acad. Sci. USA 111, 6672–6677 (2014).
61. Mergaert, P. et al. Eukaryotic control on bacterial cell cycle and
differentiation in the Rhizobium–legume symbiosis. Proc. Natl Acad. Sci.
USA 103, 5230–5235 (2006).
62. Koren, S. et al. Canu: scalable and accurate long-read assembly via adaptive
k-mer weighting and repeat separation. Genome Res. 27, 722–736 (2017).
63. Arratia, R., Lander, E. S., Tavaré, S. & Waterman, M. S. Genomic mapping
by anchoring random clones: a mathematical analysis. Genomics 11,
806–827 (1991).
64. Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial
variant detection and genome assembly improvement. PLoS ONE 9,
e112963 (2014).
65. Boetzer, M., Henkel, C. V., Jansen, H. J., Butler, D. & Pirovano, W.
Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27,
578–579 (2011).
Nature Plants | VOL 6 | February 2020 | 107–118 | www.nature.com/natureplants
Articles
66. Haas, B. J. et al. De novo transcript sequence reconstruction from RNA-seq
using the Trinity platform for reference generation and analysis. Nat. Protoc.
8, 1494–1512 (2013).
67. Benson, G. Tandem repeats finder: a program to analyze DNA sequences.
Nucleic Acids Res. 27, 573–580 (1999).
68. Tarailo-Graovac, M. & Chen, N. Using RepeatMasker to identify repetitive
elements in genomic sequences. Curr. Protoc. Bioinformatics 25,
4.10.1–4.10.14 (2009).
69. Jurka, J. et al. Repbase update, a database of eukaryotic repetitive elements.
Cytogenet. Genome Res. 110, 462–467 (2005).
70. Xu, Z. & Wang, H. LTR_FINDER: an efficient tool for the prediction of
full-length LTR retrotransposons. Nucleic Acids Res. 35, W265–W268 (2007).
71. Edgar, R. C. & Myers, E. W. PILER: identification and classification of
genomic repeats. Bioinformatics 21(Suppl. 1), i152–i158 (2005).
72. Price, A. L., Jones, N. C. & Pevzner, P. A. De novo identification of repeat
families in large genomes. Bioinformatics 21(Suppl. 1), i351–i358 (2005).
73. Stanke, M. et al. AUGUSTUS: ab initio prediction of alternative transcripts.
Nucleic Acids Res. 34, W435–W439 (2006).
74. Majoros, W. H., Pertea, M. & Salzberg, S. L. TigrScan and GlimmerHMM:
two open source ab initio eukaryotic gene-finders. Bioinformatics 20,
2878–2879 (2004).
75. Birney, E., Clamp, M. & Durbin, R. GeneWise and Genomewise.
Genome Res. 14, 988–995 (2004).
76. Holt, C. & Yandell, M. MAKER2: an annotation pipeline and genome-
database management tool for second-generation genome projects.
BMC Bioinf. 12, 491 (2011).
77. Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice
junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009).
78. Trapnell, C. et al. Differential gene and transcript expression analysis
of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7,
562–578 (2012).
79. Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinf. 10,
421 (2009).
80. Boeckmann, B. et al. The SWISS-PROT protein knowledgebase and its
supplement TrEMBL in 2003. Nucleic Acids Res. 31, 365–370 (2003).
81. Finn, R. D. et al. Pfam: the protein families database. Nucleic Acids Res. 42,
D222–D230 (2014).
82. Ashburner, M. et al. Gene ontology: tool for the unification of biology.
Nat. Genet. 25, 25–29 (2000).
83. Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes.
Nucleic Acids Res. 28, 27–30 (2000).
84. Wen, M., Shen, Y., Shi, S. & Tang, T. miREvo: an integrative microRNA
evolutionary analysis platform for next-generation sequencing experiments.
BMC Bioinf. 13, 140 (2012).
85. Lowe, T. M. & Eddy, S. R. tRNAscan-SE: a program for improved
detection of transfer RNA genes in genomic sequence. Nucleic Acids Res.
25, 955–964 (1997).
86. Nawrocki, E. P. & Eddy, S. R. Infernal 1.1: 100-fold faster RNA homology
searches. Bioinformatics 29, 2933–2935 (2013).
87. Li, L., Stoeckert, C. J. & Roos, D. S. OrthoMCL: identification of ortholog
groups for eukaryotic genomes. Genome Res. 13, 2178–2189 (2003).
88. Katoh, K., Kuma, K. I., Toh, H. & Miyata, T. MAFFT version 5:
improvement in accuracy of multiple sequence alignment. Nucleic Acids
Res. 33, 511–518 (2005).
89. Abascal, F., Zardoya, R. & Telford, M. J. TranslatorX: multiple alignment of
nucleotide sequences guided by amino acid translations. Nucleic Acids Res.
38, W7–W13 (2010).
90. Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic
analyses with thousands of taxa and mixed models. Bioinformatics 22,
2688–2690 (2006).
91. Forterre, P. & Philippe, H. Where is the root or the universal tree of life?
Bioessays 21, 871–879 (1999).
92. Matasci, N. et al. Data access for the 1,000 Plants (1KP) project. Gigascience
3, 17 (2014).
93. Yang, Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol.
Evol. 24, 1586–1591 (2007).
94. Felsenstein, J. PHYLIP: phylogenetic inference program v.3.6
(Univ. of Washington, 2005).
95. Zwaenepoel, A. & Van de Peer, Y. wgd—simple command line tools for the
analysis of ancient whole genome duplications. Bioinformatics 35,
2153–2155 (2018).
96. Van Bel, M. et al. PLAZA 4.0: an integrative resource for functional,
evolutionary and comparative plant genomics. Nucleic Acids Res. 46,
D1190–D1196 (2018).
97. Proost, S. et al. i-ADHoRe 3.0―fast and sensitive detection of genomic
homology in extremely large data sets. Nucleic Acids Res. 40, e11 (2012).
98. Zheng, Y. et al. iTAK: a program for genome-wide prediction and
classification of plant transcription factors, transcriptional regulators, and
protein kinases. Mol. Plant 9, 1667–1670 (2016).
117
Articles
99. Maddison, W. P. & Maddison, D. R. Mesquite: a modular system for
evolutionary analysis v.2.75 (Mesquite Project, 2011).
100. Madera, M. & Gough, J. A comparison of profile hidden Markov model
procedures for remote homology detection. Nucleic Acids Res. 30,
4321–4328 (2002).
101. Stamatakis, A., Hoover, P. & Rougemont, J. A rapid bootstrap algorithm for
the RAxML Web servers. Syst. Biol. 57, 758–771 (2008).
102. Sturn, A., Quackenbush, J. & Trajanoski, Z. Genesis: cluster analysis of
microarray data. Bioinformatics 18, 207–208 (2002).
103. Martens, C., Vandepoele, K. & Van de Peer, Y. Whole-genome analysis
reveals molecular innovations and evolutionary transitions in
chromalveolate species. Proc. Natl Acad. Sci. USA 105, 3427–3432 (2008).
104. Hanada, K. et al. Importance of lineage-specific expansion of plant tandem
duplicates in the adaptive response to environmental stimuli. Plant Physiol.
148, 993–1003 (2008).
105. Angellotti, M. C., Bhuiyan, S. B., Chen, G., Wan, X. & Wan, X. CodonO:
codon usage bias analysis within and across genomes. Nucleic Acids Res. 35,
W132–W136 (2007).
Acknowledgements
We thank P. R. Crane, S. Ge, D.-Y. Hong, J.-L. Huang, J.-J. Qin, Y.-L. Qiu, J.-C. Villarreal,
T. Wan and X.-Q. Wang for useful advice and discussions and L. Zhang for providing
plant pictures. We dedicate the paper to Yang Zhong in memory of his support and
valuable suggestions on this project. This work was supported by Sino–Africa Joint
Research Center, Chinese Academy of Sciences, CAS International Research and
Education Development Program (SAJC201613), the Strategic Priority Research
Program of the Chinese Academy of Sciences (XDB31000000 and XDA19050103),
National Natural Science Foundation of China (NNSF 31590822), Shenzhen Fairy
Lake Botanical Garden, State Key Laboratory of Systematic and Evolutionary Botany,
Institute of Botany, Chinese Academy of Sciences, and Key Laboratory of National
Forestry and Grassland Administration for Orchid Conservation and Utilization at
College of Landscape Architecture, Fujian Agriculture and Forestry University. Y.V.d.P.
acknowledges support from the European Union Seventh Framework Programme
(FP7/2007-2013) under European Research Council Advanced Grant Agreement
322739–DOUBLEUP.
Author contributions
Z.-D.C., Z.-J.L., Y.V.d.P. and S.-Z.Z. conceived the paper; Z.-D.C., Z.-J.L. and S.-Z.Z.
managed the project; J.Z., X.-X.F., Y.L., Z.-J.L., A.Z., Y.V.d.P. and Z.-D.C. wrote the
118
NATURe PlAnTs
manuscript; R.-Q.L., J.-F.Y., Y.-Y.L., Q.-H.W., S.-Z.Z. and M.-Z.W. collected and cultured
the plant material; R.-Q.L. and M.-H.L. sequenced and processed the raw data; X.Z. and
Z.-W.W. assembled and annotated the genome; Y.L. and J.Z. performed phylogenetic
analysis; J.Z. and X.-X.F. analysed gene families; X.-X.F. and J.Z. identified HGT; A.Z.
and Y.V.d.P. conducted WGD analysis; Y.-L. Guan. conducted DAPI staining analysis;
J.-Y.X. conducted codon-usage bias analysis; M.-H.L., G.-Q.Z. and J.-Y.W. conducted
transcriptome sequencing and analysis; S.-S.D. and Y.L. conducted the RNA-editing-site
analysis in organellar genomes; H.M., Q.-F.W., B.G., Y.J., Y.-N.J., Y.-L.Guo, H.-Z.K.,
A.-M.L. and H.-M.Y. contributed substantially to revisions. All authors commented
on the manuscript.
Competing interests
The authors declare no competing financial interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41477-019-0588-4.
Correspondence and requests for materials should be addressed to Z.-D.C., Z.-J.L.,
Y.V.d.P. or S.-Z.Z.
Peer review information Nature Plants thanks Burkhard Becker and the other,
anonymous, reviewers for their contribution to the peer review of this work.
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© The Author(s) 2020
Nature Plants | VOL 6 | February 2020 | 107–118 | www.nature.com/natureplants
 nature research | reporting summary
Zhi-Duan Chen, Zhong-Jian Liu, Yves Van de
Corresponding author(s): Peer, and Shou-Zhou Zhang
Last updated by author(s): Dec 17, 2019

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The A. angustus genome project has been deposited at the NCBI under the BioProject number PRJNA543716. The genome sequencing data were deposited in the
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Sample size We sequenced a single hornwort plant, and no statistical methods were used to predetermine sample sizes. For comparative genome
analyses, the gene sequences of Anthoceros angustus and other 18 plant species were used (Supplementary Table 13), including seven
angiosperms (Arabidopsis thaliana, Genlisea aurea, Vitis vinifera, Oryza sativa, Phalaenopsis equestris, Zostera marina and Amborella
trichopoda), one gymnosperm (Picea abies), one lycophyte (Selaginella moellendorffii), three bryophytes (Physcomitrella patens, Marchantia
polymorpha and Anthoceros angustus), two charophytes (Chara braunii and Klebsormidium nitens), five chlorophytes (Volvox carteri,
Chlamydomonas reinhardtii, Ulva mutabilis, Coccomyxa subellipsoidea and Chlorella variabilis). This sampling covered all the major lineages of
green plants and could present the backbone of green plant evolution.

Data exclusions Lines 416-456, 466-472: The prokaryotic sequences and organellar sequences were removed from sequencing data and pre-assembled
genome data. There are prokaryotic sequences and organellar sequences that involved in the genome sequencing data. Exclusion of the
contamination from foreign DNA sequences and organellar sequences is the prerequisite for accurate genome assembly. Through choose of
high-abundance k-mer reads, error-correction and MEGABLAST check, 3,78 Gb high-quality clean reads of Nanopore sequencing remained for
A. angustus genome assembly.
Lines 521-522: We excluded annotations only characterized as hypothetical/predicted protein, since these proteins could not be treated as
really functionally annotated ones.
Lines 542-543: During the comparative analysis, we chose the longest transcript to represent each gene and removed mitochondrial and
chloroplast genes, since the used genome datasets include multiple transcripts and organellar genes that might complicate the comparative
analysis.
Lines 617-618: The mean gene family size was calculated for all gene families, excluding orphans and species-specific families, since these
genes are unique to individual species and do not have orthologs in other species for comparison.
Lines 624-627: During the gene family expansion identification, transposon-derived gene families were removed, since the distribution of such
families is likely to be a consequence of the gene models derived from a repeat-masked genome sequence and therefore may be artefactual.
Lines 638-640: The sequences without support of transcript evidence were excluded from the HGT candidates, since these sequences might
be contaminated ones but not real HGT genes.

Replication The spore germination experiment was repeated three times independently. The DAPI staining experiment was repeated three times
independently.

Randomization We picked up spores randomly for germination experiments. We selected regions of the gametophytes randomly for DAPI staining.

Blinding We sequenced a single hornwort plant, and no control group is referred here. Blinding is not applicable in this study.

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